Site-directed mutagenesis of the anabaena sp. strain PCC 7120 nitrogenase active site to increase photobiological hydrogen production

摘要

Cyanobacteria use sunlight and water to produce hydrogen gas (H _2), which is potentially useful as a clean and renewable biofuel. Photobiological H_2arises primarily as an inevitable by-product of N_2 fixation by nitrogenase, an oxygen-labileenzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H_2 production by this strain, six nitrogenase amino acid residues predicted to reside within 5 ? of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N_2 Most of the 49 variants examined were deficient in N_2-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N_2 atmosphere significantly increased their in vivo rates of H_2 production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H_2 compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H_2 in an aerobic, nitrogen-containing evironment.